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Abstract

Background

Long chain omega-3 fatty acids from fish oils (O3) are known to have beneficial effects
on a number of vascular risk factors in at-risk populations. The effects of a highly
bioavailable emulsified preparation on an overweight young adult population are less
well known.

Results

Red cell and plasma phospholipid eicosapentaenoic acid and docosahexaenoic acid concentrations
increased over the four weeks of dosing in the O3 group. Dosing with O3 did not affect
central pulse wave velocity, augmentation index, or aortic systolic blood pressure.
None of the five American Heart Association metabolic syndrome components improved
over the dosing period. None of the inflammatory cytokines, C-reactive protein, or
lipids (total or LDL cholesterol) improved over the dosing period.

Conclusions

No salutary effects of O3 were observed in hemodynamic, metabolic syndrome criteria
or inflammatory markers as a result of this relatively short period of administration
in this relatively overweight, but healthy young adult cohort.

Keywords:

Background

Metabolic syndrome has become highly prevalent in the U.S. and has a strong impact
on the development of future vascular ailments including Type II diabetes mellitus
and cardiovascular diseases. This increase can be partially attributed to recent changes
in the American diet that negatively affect body weight and arterial health [1].

Arterial stiffness is associated with metabolic syndrome and is a predictor of cardiovascular
events [2]. Pulse wave velocity (PWV) and augmentation index (AIx) are measurements of arterial
distensibility [3]. Reducing dietary saturated fats and increasing omega-3 polyunsaturated fats, especially
from fish (O3), have long been known to improve vascular health and may improve measures
of arterial stiffness [4]. Dangardt et al. reported supplementation with O3s improved vascular function and
lowered the severity of inflammation among the obese [5]. However, Mackay, et al. found fish oil supplementation among those with or at risk
of heart disease and receiving aspirin and statin therapy had no effect on pulse wave
velocity [6]. O3s have also been found to improve obesity-induced metabolic syndrome through regulating
chronic inflammation [7]. These include C-reactive protein (CRP), interleukin-6 (IL-6), and tumor-necrosis
factor-α (TNF-α) [8].

While previous researchers have focused their studies on disease risk factors in older
at-risk populations [9,10], the present study sought to investigate the effects of O3 supplementation in a relatively
overweight college-aged population. The pathologies of vascular diseases have been
observed and recorded in the very young. Earlier intervention in those at early risk
may show greater promise in longer-term benefits. Researchers have found that younger
populations have responded favorably to O3 dosing during weight loss in reducing triglycerides,
leptin, insulin, insulin resistance, and blood pressure and increasing ghrelin concentrations
[11-14]. In addition, younger adults tend to consume relatively low amounts of fish and have
relatively low levels of O3s in blood and tissues [15]. This might make this group especially responsive to the beneficial effects of even
low doses of O3. Others have also found high PWV and Aix values among obese adolescents
[16]. Thus, this study investigated whether O3s improved arterial health and inflammatory
responses, both being indicators of risk for metabolic syndrome and ultimately, heart
disease, diabetes, and stroke, in a healthy young relatively overweight population.

Methods

Subjects

Healthy 18–30 year old men and women were recruited from the Appalachian State University
student population. 430 responded. Respondents were sent a survey to determine inclusion
and exclusion criteria, including self-reported height and weight and weekly exercise
engagement. Of those initial surveys, 343 were returned. Sixty subjects were invited
to participate in the study based on the following inclusion criteria: body mass index
(BMI) >23 and engagement in regular exercise of no more than 3 times per week. Subjects
were also not taking any cardiovascular medications or fish oil supplements, not allergic
to fish, or with a history of diabetes, heart disease or a stroke, and did not consume
fish more than twice per week. The research protocol was approved by the Institutional
Review Board of Appalachian State University.

Study design

The initial treatment and control groups consisted of 30 and 27 participants, respectively.
Three subjects who were invited to enter the study failed to attend the first clinical
visit. Randomization was performed from a random digits table. The subjects recruited
for this four-week, double-blind study completed two visits to the clinic. At the
first clinical visit, anthropometric measurements, including height, weight, and waist
circumference, and seated blood pressure were measured by a single trained technician.
Hemodynamic testing included central PWV and AIx, which is further used to determine
aortic pressure [17]. Hemodynamic testing was performed by 3 different trained technicians. A fasting
blood sample was drawn for blood lipids, red blood cell and plasma phospholipid fatty
acid profiles, high sensitivity C-reactive protein (hsCRP), blood glucose, and inflammatory
cytokines. A Health History Questionnaire was completed at home and returned within
the first week. Participants were requested to comply with their reported usual exercise
habits of three or less times per week during the study period.

At the conclusion of the first clinical visit, subjects were supplied with four weeks’
worth of O3 or placebo oil single-dose packets. Treatment consisted of 4 weeks of
supplementation with O3, 350 mg eicosapentaenoic acid (EPA) and 230 mg docosahexaenoic
acid (DHA) per single-dose packet, while the placebo was 1.0 g of safflower oil per
packet. The oils were emulsified products (Coromega, Inc., Vista, CA) provided in
three premeasured packets; one packet to be opened and consumed at breakfast, two
packets at dinner. Protocol compliance was monitored with a check sheet that participants
kept to record the supplements they consumed. A second clinical visit occurred at
the end of the intervention period of four weeks and included a second round of clinical
measures, blood draws and hemodynamic testing as before.

Measurements

Height and weight were determined using a Healthometer (Jarden Corp, Rye, NY) stadiometer
and scale. Waist circumference was measured manually with a tape measure. Blood pressure
was measured using a manual sphygmometer according to AHA guidelines. Blood lipids,
glucose, and CRP were measured at the local hospital laboratory.

Hemodynamic and vascular testing was performed with a manual blood pressure cuff and
sphygmomanometer and the Sphygmacor Cardiovascular Management System. Applanation
tonometry (SphygmoCor, Inc., Sydney, Australia) was used to derive the range of central
arterial indices, including PWV and AIx [17]. The same trained technician preformed each measurement and our laboratory technicians
have an intra-class correlation coefficient of 0.96 with a coefficient of variation
(%CV) of <4.0%.

Serum lipids, glucose, and hsCRP were measured on a Dimensions RXL instrument from
Siemens, Inc. Total cholesterol was measured by polychromatic endpoint technique using
oxidase, peroxidase and esterase with a%CV of 1.82%. HDL cholesterol was measured
by the accelerator selective detection method (direct measure polymer-polyanion) with
a 0.92%CV. Triglycerides were measured by enzyme immunoassay with bichromatic endpoint
with 3.60%CV. LDL cholesterol was measured by direct measure enzyme immunoassay with
bichromatic endpoint with 2.72%CV. Glucose was measured by hexokinase with bichromatic
endpoint with 3.50%CV. Finally, hsCRP was measured with colorimetric immunoassay with
bichromatic endpoint with 2.1%CV.

Levels of inflammation protein targets were measured using the Meso Scale Discovery®
Multi-Spot® Assay System. In this multiplex electrochemiluminescent ELISA, IL-6, IL-8,
IL-10, and TNF-α concentrations were detected on spatially distinct spots in single
wells on 96-well plates. Sample analyses were performed in duplicate. Intra-assay
coefficient of variability between assay plates was 11% for IL-6, 5% for IL-8, 8%
for IL-10, and 7% for TNF-α.

Fatty acid analysis was performed essentially according to the method of Lands, et
al [18]. Briefly, sample lipids were extracted by the method of Bligh and Dyer [19]. Lipid fractions were separated on thin layer chromatography. Appropriate samples
were then transesterified with boron trifluoride and extracted. Methyl esters were
separated and quantified with a Shimadzu capillary gas chromatograph with flame-ionization
detection. Authentic standards and internal standards were used. The %CVs for the
long-chain omega-3 fatty acids were under 5%.

Statistical analysis

Repeated measures general linear models were used to determine treatment effects.
Chi Square and Student t-tests were used to determine differences between treatment
groups at baseline. Analysis was performed with SPSS v.18 (SPSS, Inc. Chicago, Illinois).
A power calculation was made to estimate group size a priori. A total of 30 subjects were required to give us adequate statistical power at a
p < 0.05 for the outcome variables of PWV and AIx. Based on existing data from our
laboratory, the estimated sample size of 30 subjects gives us an effect size of 0.92
and 0.88, respectively, with an alpha set at 0.05. Test-retest reliabilities in our
laboratory for these 2 dependent variables are 0.98 and 0.97, respectively.

Results

Of the 57 men and women who entered the study, 51 completed the intervention. Six
participants did not return for the second clinical visit, four from the O3 group
and two from the placebo group. Of those that completed the study, 41 returned the
compliance check sheets, 19 from the placebo group and 22 from the fish oil group.
Of those, 100% of the placebo participants consumed greater than 85% of their supplements
while 86% of the fish oil participants consumed greater than 85% of their supplements.

Anthropometric measurements and blood samples were collected during the initial clinical
visit. Table 1 summarizes baseline information collected. Subjects chosen were at risk of metabolic
syndrome with blood pressure measurements and waist circumferences slightly higher
than normal, particularly for women. There were no statistically significant differences
in baseline measurements between the treatment groups.

Measurements were also taken four weeks later, after O3 supplementation. Figure 1 shows the effect of O3 dosing for four weeks on plasma phospholipid EPA and DHA.
Plasma levels increased significantly (p < 0.001).

Figure 1.Effect of fish oil supplementation on plasma phospholipid combined EPA and DHA levels
as percent of total fatty acids before and after treatment.

Plasma and red blood cell phospholipid fatty acid outcomes are displayed in Table 2. Significant increases are noted for both EPA and DHA and their combination in both
samples. In the plasma the combined EPA and DHA increased about 80% and in the red
blood cells the combined fatty acids increased about half as much.

The effects of supplementation on components of the metabolic syndrome, plasma lipids,
vascular measures, and cytokines are shown in Table 3. The daily treatment dose of 1.7 g of O3 for four weeks had no effect on any of these
measures compared to the safflower placebo. Not shown are BMI, waist, total cholesterol,
serum glucose, IL-8, and IL-10, all of which also showed no effect.

Discussion

In this study we investigated the effect of O3 on relatively overweight but healthy
young adults. We found no effect of 4 weeks of dosing with 1.7 grams per day on arterial
hemodynamic measures, components of the metabolic syndrome, serum lipids, or measures
of chronic inflammation. Yet, we were able to verify a change in O3 status in serum
and red cell phospholipids with O3 dosing.

Emulsifying fish oils can enhance digestion and absorption of the fatty acids. Raatz,
et al. investigated emulsified fish oil absorption compared with capsular triglyceride
fish oil supplements in humans throughout a 48-hour observation period. A single dose
(350 mg EPA and 230 mg DHA) of the emulsified product resulted in enhanced absorption
of total O3 compared with the capsular supplement. Although we gave a relatively low
dose, Raatz, et al. have shown that this material is absorbed more quickly and maybe
more completely than fish oil in tablets [20]. Figure 1 shows that the O3 treatment increased plasma O3 EPA and DHA, despite four apparent
non responders in the treatment group and two with increased EPA and DHA in the placebo
group.

Arterial stiffness is associated with metabolic syndrome [2]. PWV and pulse pressure are measures positively associated with aortic stiffening,
also measured by AIx [3,21]. Sjoberg, et al. introduced 2 g, 4 g, and 6 g of fish oil supplementation per day
into the diets of overweight or obese adults for 12 weeks. Improvement in arterial
distensibility, as measured by PWV, was only found to be significant only at the highest
dose of 6 g of fish oil per day [22]. Chong, et al. reported a significant improvement in PWV and AIx, in healthy adults
immediately after a long chain O3 PUFA-rich meal containing 4.7 g of DHA and EPA [4]. However, Sanders, et al. recently found that 1.8 g of EPA and DHA daily over 12 months
did not improve arterial stiffness among slightly overweight but relatively healthy
middle aged subjects in England [23]. Thus, using a comparable dose over a greater duration than in our study within an
older age group yielded similar outcomes.

A number of studies have been conducted on the association between O3 intake and the
development of the metabolic syndrome. The present study found that fish oils did
not have an effect on components of the metabolic syndrome in overweight young adults.
Pederson, et al. found that supplementation with 1.5 g of O3 per day for 16 weeks
significantly lowered systolic blood pressure and raised HDL cholesterol in overweight
adolescents [24]. Also, supplementation with O3 over a 12-week period significantly lowered serum
glucose levels [25].

Inflammation is also recognized as having a significant relationship with metabolic
syndrome [26]. Dietary patterns poor in O3 may cause an excessive production of pro-inflammatory
cytokines and CRP, while causing a lower production of anti-inflammatory cytokines,
all recognized as contributing to the inflammation associated with metabolic syndrome
and cardiovascular events [26,27]. Dangardt, et al. executed an intervention of 1.2 g of O3 supplements per day on
obese adolescents for 3 months having an average BMI of 33.8, compared to our group
average BMI of 28.1. Results showed a significant decrease in TNF-α and IL-6 levels,
but no significant change in the serum levels of CRP, IL-8 or IL-10 [5]. Low CRP levels have also been observed in Yup’ik Eskimos, a population who have
mean daily intakes of DHA and EPA ranging from 2.4 to 3.7 g. In this population, CRP
blood levels were inversely related to the intake of fish oils; however, there was
no relationship found with IL-6 levels and fish oil intake [28]. Our results, in addition to those found in past studies, may further demonstrate
the need for a longer intervention period, a higher treatment dose and an at-risk
population selection for treatment to observe the desired results in inflammatory
markers.

Overall, our present investigation found little effect of emulsified fish oil on components
of the metabolic syndrome, inflammatory cytokines, or hemodynamic measures of arterial
health. Other studies suggest that higher doses of fish oil coupled with a longer
intervention period executed in a more unhealthy population may be needed to manifest
positive and significant results from O3 intervention. Strengths of our study include
the bioavailability of the emulsified supplement, clear evidence for incorporation
of O3 doses into blood phospholipids, the young age of the participants, and the multiplicity
of the endpoints, specifically the hemodynamic markers and the inflammatory cytokines.
However, some limitations to the study are apparent. These include the short length
in time of the dosing and the relatively low dose compared to other more recent studies.

Conclusions

The administration of a highly bioavailable emulsified preparation of fish oil O3
measurably increased plasma and red blood cells EPA and DHA in four weeks. However,
no beneficial changes were observed in the markers that were measured for hemodynamic,
metabolic syndrome, or inflammatory effects as a result in this relatively healthy
overweight young adult cohort. More at-risk populations may benefit more from this
intervention.

Competing interests

Martin Root received funding for this research from the Dyson Foundation and the supplements
from Coromega, Inc. The other authors declare that they have no competing interests.

Authors’ contributions

MR was the Principal Investigator. He directed the study, the data analysis and the
drafting of the manuscript. He finalized and submitted the manuscript. SC directed
the arterial function testing and data interpretation and hosted study site. KZ conducted
the inflammatory marker analysis and data interpretation. KW wrote the initial manuscript
and performed the data analysis. MM managed the trial, recruiting subjects, scheduling
both subjects and researchers and coordinating data collection. All authors reviewed
and contributed to the final version of the manuscript.

References

Committee DGA: Part D. Section 3: Fatty Acids and Cholesterol.

Report of the Dietary Guidelines Advisory Committee on the Dietary Guidelines for
Americans, 2010 2010.